Bio355

1. Biochemical Activities of Microorganisms
Sercan Güloğlu
19-21 December 2018

2. Voges Proskauer
1. Take 1 mL to a new tube.
2. Add 0,5 mL Barrit’s A solution.
3. Add 0,3 mL Barrit’s B solution.
4. Shake vigorously every 20 sec for a minute. Wait for 1-2 hours for the results.
• Red Color: acethylmethyl carbinol presence which means the production
of 2,3 butanediol from pyruvic acid.
2
Indole Methylred Voges-proskauer i Citrate

3. 3
Methyl Red
1. Take 2 mL to a new tube
2. Add 5 drops of methyl red
• Red color: Acidic. (Mixed acid production
• Yellow color: Alkaline (No mixed acid production).
Indole Methylred Voges-proskauer i Citrate

4. Gelatin Hydrolysis
• Put your tubes into the fridge.
• Make sure they are melt before putting.
4
Nitrogen Metabolism

5. Carbon Metabolism
Starch Hydrolysis
Flood surface of the agar with iodine
Dark blue : No amylase activity.
Clear zone around colonies: Alpha & Beta amylase activity.
Red complex : Alpha amylase activity only (amylopectin remains intact).
5

6. Carbon Metabolism
6
Phenol Red Broth (Glucose & Lactose)
1. Check the bacteria growth (do not mix the tube) (maybe in a form of precipitation)
2. Check if there is any bubble in the Durham tube (Gas production)
3. Check the color of the broth:
– Yellow: Acidic
– Red: Alkaline

7. 7
Indole Production (Tryptone broth)
1. Check the bacterial growth.
2. Add 10 drops of Kovac’s reagent and mix well in your hand.
• Red Color: Indole production.
Nitrogen Metabolism

8. Urea Utilization
1. Check bacterial growth.
2. Observe the color of the slant.
• Pink-Red Color: Urease activity. NH3 production increased the pH.
• Yellow Color: No urease activity.
8
Nitrogen Metabolism

9. Gelatin Hydrolysis
Get your tubes from the fridge.
1. Observe vertical growth of bacteria (growth control).
2. Observe liquefaction by holding tube inclined.
– Liquid: Gelatinase activity.
– No liquid: No gelatinase activity.
9
Nitrogen Metabolism

10. Nitrate Reduction
1. Check bacterial growth.
2. Check the bubble formation in the Durham tube.
• Gas production: All enzymes are active.
• No gas production: Not all enzymes are active.
10
Nitrogen Metabolism
3. Take 2 mL to a clean test tube.
4. 2 drops of Sulfanilic acid then 1 drop dimethyl-alpha-naphtylamine(for nitrite detection).
• Red color: Reduction of nitrate into nitrite.
• No color: There is no nitrite in the medium.

11. Nitrate Reduction(cont’d)
5. Add a pinch of zinc dust to your original culture.
• Red color: Unreduced Nitrate present in the culture.
• No color: Nitrate is reduced (Nitrate reductase is active).
11
Nitrogen Metabolism

12. 12
Indole Methylred Voges-proskauer i Citrate
Indole Production (Tryptone broth)
Use the data from the indole production test previously observed.

13. Citrate Utilization
1. Observe the color change.
• Blue: Citrate utilization as a carbon source.
• Green: Citrate is not a Carbon source for corresponding organism.
13
Indole Methylred Voges-proskauer i Citrate

14. 14
Litmus Milk
1. Check the acid curd formation (insoluble complex of calcium and casein).
• Purple to pink (curd formation).
2. Check for alkaline reaction.
• Blue: Alkaline formation.
• Purple: No alkaline formation.
Multiple Test Media

15. 15
Litmus Milk (cont’d)
3. Check for the peptonization (digestion of milk protein casein by proteases).
• Reduction in curd size and formation of brownish liquid.
4. Check for litmus color change.
• White color: reduction of litmus.
Multiple Test Media

16. 16
Litmus Milk (cont’d)
5. Check for the gas formation (furrow formation in the curd).
• Furrows: gas production.
6. Check for ropiness (Immerse a loop into the medium and slowly remove it.
Observe if there is any string).
• String: indication of ropiness.
Multiple Test Media

17. 17
Triple Sugar Iron (TSIA)
1. Check for the colors of slant and butt seperately.
• Yellow butt: glucose utilization.
• Red color on the slant: only glucose fermenters.
• Yellow slant: organism can also ferment lactose and/or sucrose.
2. Check for the gas formation.
3. Check for the H2S formation(detected by FeS).
• Black colored agar: H2S formation.
Multiple Test Media

18. Sulfide Indole Motility (SIM)
1. Check for the color of your culture.
• Dark color: hydrogen sulfide production.
2. Check for the diffusion of the colonies.
• Diffused color: motile organism.
3. Add 10 drops of Kovac’s reagent. Observe the color change.
• Pink color: Indole production.
18
Multiple Test Media

19. 19
REPORTS

20. 20
Positive outcome:
Our starch agar experiment results showed that our organism can hydrolyse the starch. Upon
addition of iodine on the agar, clearing occured as whitish color around the colonies which is an
indicator of both alpha and beta amylase activity. It has been previously shown that Bacillus subtilis
gives positive reaction clearing colony around [1,2]. Therefore, this outcome supports that our
unknown bacterium might be Bacillus subtilis.
Negative outcome
Our starch agar experiment results showed that our organism cannot hydrolyse the starch. Upon
addition of iodine on the agar, nothing changed around the colonies which is an indicator of no
amylase activity. It has been previously shown that Escherichia coli gives negative reaction on
starch agar test [3]. Therefore, this outcome supports that our unknown bacterium might be
Escherichia coli.
In-text reference
In-text reference

21. 21
• 4 pages (maximum) of reports will be arranged as follows:
• Cover page: Your section and group number. Write your group members as well.
• Maximum 2 pages of discussion (1 page is preferred).
• 1 separate page for the reference list. (Check for the reference tools for correct reference formatting).
• Font: Arial---10 puncta---Centered text---Word format
• Mail your reports to: guloglu.sercann@gmail.com
• No questions about any test’s evaluation will be answered !!!
• You can use websites as a reference. However, scientific article references will receive extra points.
• You have 7 days for report submission.
• Earlier report submissions will get you extra 5 points per day up to 4 days (+20 points in total)
• Later report submissions will take away your 10 points per day.

22. 22
• Grading of the reports:
Points
Cover page 5
Introduction pragraph (2-3 sentences) 5.4
Evaluation of 11 different tests
(11 separate paragraphs)
5.4 (each paragraph)
59 (total)
Conclusion paragraph 5.4
References
(correct formatting & correct information)
10
Language & text formatting 5
Correct prediction of the culture 10
Total 100
Scientific article reference +2 (per article)
Earlier report submission
+5 (per day)
+20 (maximum)
Later report submission -10 (per day)